They should grow. But there are many reasons why plating fails. Did you see this paper?

Article Development of a Solid Medium for Growth and Isolation of Ax...

Maybe the medium mentioned in there is better? Also compare your light intensity and your incubation temperature with that in this paper.

Another idea: Did you use streak-plating? If not, do it. Because you will have a lot of cells in one corner of your plate and they typically become visible as a green lawn within a few days. This provides a good control.

I would consult., "Standard Methods for the Examination of water and Waste water" available on line as well as from "American Water Works Association".

Dear Tangjian Peng ,

We are working on this issue with M. aeruginosa at this moment at our lab within our current project. We culture them in 0.5X BG-11, and for solid media we add 15.0 g/ L of Bacteriological Agar and they grow quite well.

There are not many studies reporting 100% axenic cultures of M. aeruginosa, since it seems quite difficult to accomplish it. This is due to the fact that many heterotrophic bacteria are tightly attached to M. aeruginosa being part of its microbiome.

We started using as guide the methods reported by these two studies:

• Han, A. W., Oh, K. H., Jheong, W. H., & Cho, Y. C. (2010). Establishment of an axenic culture of microcystin-producing Microcystis aeruginosa isolated from a Korean reservoir. Journal of microbiology and biotechnology, 20(7), 1152-1155.
• Harke, M. J., Berry, D. L., Ammerman, J. W., & Gobler, C. J. (2012). Molecular response of the bloom-forming cyanobacterium, Microcystis aeruginosa, to phosphorus limitation. Microbial ecology, 63(1), 188-198.

We are currently testing different approaches to see which is the best method for obtaining axenic lines, and I would summarize them in the following:

1) Striking is the most classical and cheap method to do it, combined with serial dilutions and alternation between solid/liquid, but it can be time consuming. We are currently trying it with 3 strains of M. aeruginosa.

2) A complementary approach to striking is the use of FACS citometry, I highly recomend you check this recent publication of Doppler et al. (2021):

Article Make microalgal cultures axenic again – a fast and simple wo...

3) Combine the use of physical cleaning (centrifugation, ultrasound and filter cleaning) with antibiotics. Physical cleaning by itself have helped us to remove excessive bacterial contamination (up to 90-92% of initial bacterial cells) and it increased up to 95-98% by using D-cicloserine (Harke et al. 2012) but 10 times the concentration reported. We have not tried Imipenem but it seems to work also well (Harke et al. 2012).

Another aspect is how you test for axenicity. Visual checking under epiflurescence microscopy combined with staining (e.g. SYBR green or DAPI) seems to works well and has been applied by numerous studies (e.g. Porter and Feig 1980; Bolch et al. 1996, Choi et al. 2007, Han et al. 2010). However, SEM microscopy would defenitely test that not bacteria remain stuck to the cell surface of M. aeruginosa (e.g. Han et al. 2010, Cho et al. 2013).

I hope this can help you explore different ways to obtain you axenic line. Do not hesitate to contact us for further questions.

Best,

Raquel

We have used an antibiotic method to produce axenic algae cultures. This might work with your one. If you talk to a microbiologist, they have a kit used to test sensitivity of bacteria to many antibiotics. Your culture is spread on agar and different paper disks containing different antibiotics are placed on the agar plate. Look for a clear zone around a paper disk where the bacteria are killed but your alga is not affected. This tells you which antibiotic to add to your culture medium. Our technique was published previously as “One step antibiotic disk method for obtaining axenic cultures of multicellular marine algae.” Bradley et al. 1988. Plant cell, tissue and organ culture 12: 55-60. Available on Peter Bradley’s ResearchGate place. Good luck with your work.

Dear, Tangjian Peng

1. you can use "Glass capillary method" to isolate and purify cyanobacterial/microalgae culture. (Best for unicellular algae)

First of all you take a sterile glass capillary tube, put it on the burning sprit lamp/ fire flame with hold it to both end of capillary then stretch it and break it into two parts and create a hair like capillary tube from the center, break end portion of capillary tube with the help of sterile forceps.

Further, take a cyanobacterial growing on agar petri dish ((1-2% agar), open it in the surrounding of fire flame and select a single cyanobacterial colony with the help of hair like end of glass capillary tube and transfer it into a fresh media (BG-11/BBM or others). Repeat it 2-3 time then you will got pure cyanobacterial/ microalgal culture.

2. you can use "Antibiotic method" (best for filamentous algae but rarely use for unicellular algae )

here, (i) first of all, cyanobacterial growing culture (on agar slant or agar petri dish) keep in light for one week.(ii) kept in dark for 72 hour (iii) add chloramphenicol (20, 40 µg/ml) and again keep in dark for 72 hour (iv) after 72 hour wash it with autoclaved double distilled water (DDW). (v) Pic a cyanobacterial colony and plating it on agar plate then you will got pure cyanobacterial/ microalgal culture.

I have attached picture of glass capillary tube below.

Best Wishes.

Dear Tangjian Peng....You can use specific antibiotic for microalgae

Hi, you can check the precedure in the Supplements of the bellowing article:

Article Colony formation in two Microcystis morphotypes: Effects of ...